The inventions described herein relate to the affinity separation and detection of analytes in a sample. In particular, they relate to reagentless binding assays which permit contemporaneous (real time) monitoring of analyte concentration, detection of analyte at very low concentrations, and which are easily generalized for detection and quantitation of a host of different analytes.
1. Field of Art
The assays of the invention employ unique modular affinity assemblies as affinity matrices for affinity separation, detection, and quantitation of analyte. The assembly includes at least one sensor unit, each comprising at least one anti-analyte receptor (affinity module) and at least one signal-competent reporter probe (reporter module) signal-responsive to events associated with analyte/receptor complex formation. While the sensor units are exemplified herein as optical sensor units comprising luminescent reporter probes (luminophors), the use of binding-sensitive reporter probes which transduce non-optical signals such as electron transfer signals such as electron transfer and radioactivity in the modular assemblies of the invention are also contemplated.
The inventions are particularly useful for the detection of very dilute concentrations of analyte (in the ng/ml-pg/ml range and lower), and for continuously reporting changing concentrations of analyte in xe2x80x9creal time,xe2x80x9d (i.e., contemporaneously with the change in concentration), without further manipulation. Readings of the transduced signal can easily be taken at locations remote from the point of analysis, thus permitting, for example, constant monitoring of environmental air and water for pollutants, from afar.
Other applications for the assays of the invention include the clinical detection and real-time monitoring of trace biochemicals in body tissues and fluids, thus permitting, for example, the diagnosis and monitoring of biochemical markers of disease; the detection of trace amounts of hazardous (bio) chemicals in the environment such as those from medical, radioactive or industrial waste; the detection of pathogens (e.g., microorganisms and viruses or their toxins) in minute quantities in the atmosphere (including closed environments such as health-care facilities) or water supply; the detection of explosive materials; and the detection of proscribed substances such as controlled drugs.
2. Discussion of Related Art
Numerous assays based upon affinity chromatography are well-known for the detection of analyte in a sample. Typically, such assays involve immobilizing an analyte-specific, labelled ligand on a support to form an affinity matrix; contacting the immobilized ligand with a fluid sample potentially containing analyte; and detecting and/or quantitating bound analyte. Competitive binding assays, dependent upon competition between analyte and a labelled analyte analog for ligand binding sites are more complicated, additionally requiring labelled analyte analogs for initial saturation of matrix-bound ligands. Sandwich-type binding assays also additionally require labelled secondary ligands for sandwiching analyte bound to the primary immobilized ligand.
These classic assays have several drawbacks which make them unsuitable for achieving the objectives of the present invention. In both assays, the equilibrium eventually established between the analyte and immobilized ligand reflects a static sample analyte concentration, but fluctuations in the concentration cannot be detected without further addition of reagents, viz., labelled analog or labelled secondary ligand. Further, the assay is not reversible without time-consuming regeneration of the matrix, and even so this is not always possible. The present invention obviates the need for secondary labelling steps that require time for mixing, reaction, and flushing of excess reagent prior to detection and quantitation of analyte, and permits real-time continuous monitoring of analyte concentration without matrix regeneration.
The development of reagentless fluorescence-based sensors has long been a goal in this art. Several reagentless techniques that allow nearly real-time monitoring of chemicals have been developed, but all have been limited to a narrow range of analytes and receptors. In many of these techniques, the receptor itself is a fluorescent molecule (e.g., a metal ion chelator) whose fluorescent properties (e.g., emission intensity, emission wavelength, or lifetime) change upon analyte binding. Accordingly, a large effort has been expended on developing fluors that also have analyte recognition properties.
For example, a variety of cation-specific fluorescent dyes are commercially available that exhibit an intensity enhancement or a shift in their fluorescence emission spectra upon ion binding,. In addition to pH sensitive dyes, there are several known fluorescent probes that specifically chelate ions such as Mg+2 (e.g., furaptra), Zn+2 (TSQ), Na+ (SBFI), K+ (PBFI), and Ca+2 (EGTA-AM) for use in the detection of relevant chemicals (Haugland, R. P., Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, 1996). These dyes have found wide application both in environmental sensing applications and in biochemical studies. The methods employed for detection of analyte with these dyes, however, have the disadvantage of requiring a specific receptor for the analyte that is also fluorescent, and whose fluorescent properties change upon analyte binding. Thus, they cannot be generalized to arbitrary analytes. Similar restrictions also apply to other optical sensor units used to detect excited state fluorophors or phosphors (Meier, B.; Werner, T.; Klimant, I.; Wolfbeis, O. S., xe2x80x9cNovel Oxygen Sensor Material Based on a Ruthenium Bipyridyl Complex Encapsulated in Zeolite-Y: Dramatic Differences in the Efficiency of Luminescence Quenching by oxygen on Going, From Surface-Adsorbed to Zeolite-Encapsulated Fluorophores,xe2x80x9d Sensors and actuators B 2: 240, 1995).
A related reagentless approach is the use of environmentally-sensitive dyes with proteins, polymers, and in molecular assemblies (Lundgren, J. S.; Bright, F. V., xe2x80x9cBiosensor for the Nonspecific Determination of Ionic Surfactants,xe2x80x9d Anal. Chem. 68: 3377, 1996). Dyes such as 6-propionyl-2-dimethylamino-naphthalene (prodan) and 6-dodecanoyl-2-dimethylamino-naphthalene (laurodan) have been used for binding of a wide range of analytes. These dyes incorporate both electron donor and electron acceptor moieties that result in a large dipole moment in the excited state (Haugland, R. P., Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, 1996). Consequently, the emission spectra of these dyes are extremely sensitive to the polarity of their environment. For example, the emission maximum for prodan varies from about 380 nm when the dye is in a nonpolar environment (e.g., cyclohexane), to about 520 nm when the dye is in a polar environment (e.g., water) (Haugland, R. P., Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, 1996). In typical sensing protocols, these dyes are incorporated into polymers that swell in the presence of certain types of low molecular weight analytes (Barnard, S. M.; Walt, D. R. xe2x80x9cA Fibre-Optic Chemical Sensor with Discrete Sensing Sites,xe2x80x9d Nature 353: 338-340, 1991). The degree of swelling of the polymer is directly proportional to the amount of analyte that has been imbibed by the polymer, and the resulting change in the polarity of the environment of the dye affects its fluorescence emission properties, which can then be correlated to analyte concentration. Similar techniques have been used to study the dynamics of cell membranes. For example, the effects of drugs, anesthetics, extracellular proteins, and metal ions on membrane dynamics and structure have been monitored by the incorporation of fluorescent probes such as diphenylhexatriene into the membrane structure (Haugland, R. P., Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, 1996). Although these general methods for fluorescence-based transduction allow reagentless monitoring of a variety of types of analytes, it is not broadly generalizable and it also lacks analyte specificity, as many background substances can cause changes in the environment of the fluorescent molecule that lead to confounding spectral data.
One type of reagentless, fluorescent sensor unit having a high degree of specificity comprises a specific binding receptor protein modified by covalent incorporation of reporter fluors. These sensor units are engineered so that emission properties of the fluor are changed with analyte/receptor binding. As an example, three general transduction mechanisms in which binding of analyte to the modified receptor results in a perturbation of the fluorescence emission have been identified (see Case et al and Sohanpal, K.; Watsuji, T.; Zhou, L. Q.; Cass, A. E. G., xe2x80x9cReagentless Fluorescence Sensors Based Upon Specific Binding Proteins,xe2x80x9d Sensors and Actuators B 11: 547, 1993). These are (1) perturbation of the fluor either through direct interaction with the analyte upon binding, or through indirect interaction via the receptor upon binding; (2) perturbation of the fluor caused by conformational changes in the receptor protein upon analyte binding that results in a change in the local chemical environment of the fluor; and (3) perturbation of the fluor caused by a change in the aggregation state of the receptor protein upon analyte binding. Although these transduction mechanisms are reagentless, reversible, and specific, they are too specific to be generalizable, as the fluor must be preattached to each protein receptor at a site that typically differs depending upon the protein receptor selected.